[Histonet] Uneven Staining

[Rene J Buesa]

This is quite a puzzling situation.
You have ruled out dewaxing (that was my first idea of cause) but I do not think that soaking will be the problem because the sections are fixed and infiltrated with paraffin, so there is no possibility of "diluting" in water.
Thick/thin sections in a series is a very rare occurrence so I am also at a loss with this problem. Weak staining solutions could cause it but all sections in a series will be equally weak.
Just in case, since you have covered the dewaxing, issue do the following: do not leave the sections too long in the water bath and dry them in the oven as soon as they are drained.
René J.

From: Elizabeth Cameron <Elizabeth.Cameron <@t> jax.org>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Tuesday, November 27, 2012 1:35 PM
Subject: [Histonet] Uneven Staining

About a year ago, I posted about issues with staining mouse kidney sections (see below for original post).  We realized that part of the problem was the sections drying during staining, which has been resolved, but we are still having some issues with some slides staining well and others being very pale.  This time it seems to be consistent across the slide, so I know it is not a drying issue.  They are serial sections, so they are all cut together and stained together, and they are from the same animal.  I don't believe deparaffinization is an issue - we have tried longer times with fresh solutions and it doesn't make a difference.  We have also tried re-staining the slides after letting them sit in xylene overnight, and there is no difference in the staining.  I am now thinking that it may be related to how long the sections soak or how long they sit on the waterbath.  They are NBF fixed, and there is a fine line between soaking well for
 hydration and oversoaking, resulting in swelling.  Has anyone experienced anything like this?  Any other ideas?  I am at a loss, and I'd really like for this to work!
Thanks,
Liz

We are doing a Hale's colloidal iron stain (no counterstain) on serial sections of mouse kidneys.  We are staining an entire kidney at a time (about 90-150 slides), and after many successful runs, we are now finding some slides in each batch with very uneven staining.  Half of a section will stain as it should, and the rest of the section is very pale.  It seems to be in a similar area from one section to the next, but not exactly the same area.  It does not look like a deparaffinization issue.  There may be two or three slides in a row like this, or just one section on a slide, followed by 30-40 that look fine.  The sections are 6 microns. Any ideas on why this might be happening?  Thanks!
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